Kudzuvine root and sesame coffee for improving non-alcoholic fatty liver and application of kudzuvine root and sesame coffee

Abstract

The invention relates to the technical field of biological medicine, in particular to radix puerariae and sesame coffee for improving non-alcoholic fatty liver and application of the radix puerariae and sesame coffee. The radix puerariae and sesame coffee comprises the following components in parts by weight: 0.5 part of a fructus cannabis polypeptide solution, 0.5 part of radix puerariae polypeptide solution and 9 parts of coffee. After continuous treatment for 7 weeks, according to liver appearance and liver histopathology observation, it is found that the radix puerariae and sesame coffee mixed liquid effectively reduces lipid deposition and liver fat, and the liver fat lesion is improved. Experimental results show that the radix puerariae and hemp coffee has a remarkable lipid-lowering effect, and can effectively reduce liver fat particles, reduce inflammatory injury, improve liver tissue pathological changes and relieve NAFLD mouse liver tissue injury.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to kudzu coffee for improving non-alcoholic fatty liver disease and application thereof. Background Art

[0002] 1.1 Non-alcoholic fatty liver disease

[0003] 1.1.1 Causes and pathogenesis of non-alcoholic fatty liver disease

[0004] According to the different causes, fatty liver is divided into alcoholic fatty liver and non-alcoholic fatty liver. Alcoholic fatty liver is caused by alcoholism and is characterized by excessive fat deposition in liver cells, accompanied by liver cell damage and necrosis. [1] The main treatment for alcoholic fatty liver disease is to stop drinking to reduce liver cell damage and improve liver function. Diet and exercise can also be adjusted to improve liver disease symptoms and reduce the risk of liver disease worsening. [2] Nonalcoholic fatty liver disease (NAFLD) is caused by excessive fat deposition in the liver due to metabolic disorders, obesity, diabetes, cardiovascular disease, inflammation and malnutrition. [3] The treatment principles for non-alcoholic fatty liver disease are to improve obesity, control blood sugar, improve diet and exercise habits, and control lipid metabolism disorders. [4] Drug treatment can also be used to improve liver disease symptoms, reduce fat deposition in liver cells, improve liver function, and reduce the risk of liver disease progression. [5] Patients with NAFLD generally do not have a history of drinking, but their liver parenchymal cells show fatty degeneration and fat accumulation. Today, NAFLD has become a global health problem. [6] , affecting approximately 24% of the world’s population [7] If not treated promptly, NAFLD may develop into cirrhosis, liver failure, or even hepatocellular carcinoma, seriously affecting quality of life and even leading to death. [8] . Therefore, it is crucial to understand the risk factors that are closely related to the development of non-alcoholic fatty liver disease, which include overweight, obesity, high blood pressure, diabetes, high cholesterol levels, dietary habits, drug abuse, lack of physical activity, birth weight and genetic factors. In addition, continuous damage to the liver from fat accumulation and inflammation may also lead to the formation of fatty liver. Insulin resistance can lead to fat accumulation, lipid metabolism disorders can lead to lipid synthesis and decomposition disorders, oxidative stress can lead to cell damage, inflammation can lead to inflammatory responses, apoptosis can lead to liver cell death, and fibrosis can lead to impaired liver function. At the same time, viral hepatitis, drug toxicity, dietary abuse, and malnutrition are also risk factors for the development of non-alcoholic fatty liver disease. [9]Regulating lipid metabolism disorders may be an effective way to treat non-alcoholic fatty liver disease. Certain drugs can be used for liver protection treatment. In addition, it is recommended to reduce weight by controlling diet and exercising to alleviate the disease.

[0005] The most widely accepted theory of pathogenesis in modern medicine is the "two-hit theory"

[10] The first blow is insulin resistance, which weakens insulin's ability to regulate fat. Due to insulin resistance, the level of free fatty acids in the blood increases, allowing them to enter the liver directly, leading to excessive accumulation of fatty acids in liver cells. It also inhibits the oxidation of fatty acids and reduces their utilization, leading to increased fatty acid accumulation in the liver, which in turn increases the synthesis of very low-density lipoproteins and reduces their degradation, ultimately leading to an increase in plasma very low-density lipoproteins, high triglycerides, and low high-density lipoproteinemia. The second blow is fat deposition and lipid peroxidation, which is caused by the presence of reactive oxygen species.

[11] Reactive oxygen is a very active substance that breaks down fats and lipids into smaller substances and stores them in the body's cells. In addition, reactive oxygen can also destroy the structure of fats and lipids, making them more toxic. These reactive oxygen species can also damage proteins and DNA in cells, leading to cell degeneration and cell death.

[12] . In addition, reactive oxygen species can also inhibit the function of the body's immune system, leading to more diseases and inflammation. Therefore, reactive oxygen species play a vital role in fat deposition and lipid peroxidation. They have strong oxidizing properties and can attack lipids and proteins in cells, aggravating fat deposition and lipid peroxidation. Superoxide anions, hydrogen peroxide, singlet oxygen, and triplet oxygen are all important components of reactive oxygen species. They are highly destructive to tissue cells, not only causing damage to proteins and DNA, but also damaging mitochondria, thereby affecting the metabolism of free fatty acids and aggravating lipid deposition in the liver. In addition, they can also destroy the structure and function of biological membranes, leading to inflammatory cells infiltrating hepatocytes.

[0006] 1.1.2 Treatment of non-alcoholic fatty liver disease

[0007] (1) Treatment with Traditional Chinese Medicine: Traditional Chinese medicine believes that the disease is caused by excessive diet, lack of exercise, physical exhaustion, etc., because the essence of water and grain is not properly gasified, leading to dysfunction of the liver, spleen, and kidney.

[13] Clinicians will use traditional Chinese medicine to treat fatty liver from multiple aspects and multiple targets, achieving good clinical efficacy. Clinicians often treat fatty liver from the perspectives of soothing the liver and promoting qi, strengthening the spleen and clearing turbidity, and warming the kidney and clearing turbidity. Non-alcoholic fatty liver disease is more common in middle-aged and elderly people.

[14] . With the acceleration of the pace of life and work, competition is becoming increasingly fierce, people are physically and mentally fatigued, overeating fatty and sweet foods, which exceeds the ability of the spleen and stomach to transport and transform, resulting in the failure of water and grain to return to normal, and eventually forming phlegm and fat. These phlegm and fat accumulate in the liver, affecting the normal operation of the liver's qi. Phlegm and qi stagnation turn into heat over time, and the liver yin is damaged. The liver and kidney are of the same origin, and the kidney's yin essence is also damaged. The clinical manifestations of patients with fatty liver are often liver failure to dredge, spleen failure to move, and kidney failure to steam. The treatment methods of soothing the liver, strengthening the spleen, and warming the kidney are often used, but taking these drugs for a long time will damage people's yin and blood. Therefore, the pathogenesis of patients with fatty liver is often accompanied by symptoms of yin deficiency.

[0008] (2) Western medicine treatment: mainly diet and physical exercise. Both methods are effective ways to improve metabolic disorders and insulin resistance (IR). Through dieting, body fat content can be reduced, thereby reducing body weight and body fat ratio, thereby improving IR. Physical exercise can change the body fat composition, and effectively improve IR by strengthening muscle and cardiopulmonary function and reducing body fat content. Diet and exercise can also improve biochemical indicators such as serum transaminase, improve liver function, lower serum cholesterol levels, and reduce inflammation, thereby improving metabolic disorders. Therefore, diet and exercise are effective ways to improve insulin resistance (IR) and metabolic disorders, and can effectively improve insulin sensitivity, thereby achieving the purpose of improving IR and metabolic disorders. Therefore, it is recommended that patients of any age should take these measures to improve their IR and metabolic disorders.

[15] Modern medicine has taken an active approach to the treatment of non-alcoholic fatty liver disease. Effective drug treatments have been adopted to address issues such as insulin resistance, lipid peroxidation, and dyslipidemia. Clinical application of HMG-CoA reductase inhibitors: pravastatin, simvastatin, fluvastatin, etc. can effectively lower blood lipids and prevent the occurrence of cardiovascular diseases.

[0009] 1.2 Hemp Seed

[0010] 1.2.1 Active ingredients of hemp seed

[0011] Hemp seed is the dried mature fruit of Cannabis sativa L., a plant of the Moraceae family. It is an annual herb and was first recorded in Shennong's Herbal Classic.

[16] . It is sweet and flat in nature, and enters the spleen, stomach, and large intestine meridians. Also known as hemp, thread hemp, industrial hemp, etc., it is a non-toxic hemp. The seeds are flat and oval, with a slightly pointed top, about 4 to 5 mm long and 2.5 to 4 mm in diameter. They are usually gray or gray-brown, with watermelon-like stripes on the surface and edges on both sides. It is a herbaceous plant of the genus Cannabis in the Cannabaceae family. It is planted in many places in Northeast my country, North China, and South China as a fiber or oil crop. The hemp stem bark can be used to weave cloth, make paper, or weave ropes; the leaves can be used as medicine. It is recorded in "Puji Fang" that it is used to treat malaria; the seeds can be pressed for oil, fried, and used as animal feed. It has a history of more than 3,000 years. Hemp seed is a nutritious food and Chinese medicinal material, rich in a large number of nutrients and active ingredients. These include a variety of vitamins, minerals, and amino acids. It is rich in unsaturated fatty acids, including linoleic acid, oleic acid, linalool, palmitic acid, and arachidic acid. There is also rich protein, with a content of about 20% to 25%, including high-quality amino acids such as glutamic acid, arginine, tryptophan, valine and threonine. In addition, there are 10% to 15% dietary fiber and many minerals, providing rich nutrition for the human body.

[17] It is reported that the protein in hemp seed is mainly globulin and albumin, with a protein content of about 20% to 25%. After removing the oil, the protein content in hemp seed meal is as high as 68.66%.

[18] . The amino acids in hemp seed protein are very rich, including many essential amino acids for the human body. Hemp seed protein contains a relatively high content of arginine and histidine, which are very important for growing children. At the same time, arginine can be converted into nitric oxide in the body, which has the effect of dilating blood vessels, lowering blood pressure, and keeping blood vessels unobstructed and elastic. Arginine can treat hypertension, hepatic coma, cirrhosis, etc. Hemp seed protein also has relatively high sulfur-containing amino acids, methionine and cysteine, which are all essential amino acids for synthetase

[19] They can not only meet the nutritional needs of the human body, but also promote the healthy development of the human body.

[20] Therefore, hemp seed protein is a high-quality plant protein resource. However, the structure of hemp seed protein is tight, and the solubility is poor after the protein is denatured after heating, which affects its digestibility. Its oral bioavailability is not ideal, so it is necessary to modify it to improve its utilization rate.

[21] Studies have shown that the use of alkali dissolution and acid precipitation to extract hemp seed protein and then hydrolyze the protein with alkaline or neutral protease can obtain two types of hemp seed peptides: antioxidant peptides and antihypertensive peptides.

[22] Hemp seed polypeptide has many functions, including anti-oxidation, lowering blood pressure, lowering blood sugar, regulating immunity, etc., and has extremely high development value.

[23] Hemp seed polypeptide is also rich in lignanamide, which can effectively inhibit inflammation and has anti-tumor and cardiovascular and cerebrovascular protection effects.

[0012] 1.2.2 Pharmacological effects of hemp seed

[0013] Hemp seeds can be processed into edible hemp seed powder, hemp seed oil, hemp seed sauce, etc.

[24] In 2002, hemp seed was listed as "both food and medicine" and included in the "Chinese Pharmacopoeia" as a nutritious and powerful health care product.

[25] It is recorded in many ancient medical books such as "Jinxiao Fang" that hemp seed has the effects of nourishing yin and replenishing deficiency, moistening the intestines and relieving constipation, aiding digestion, and treating blood deficiency and body fluid deficiency. It is also effective for diseases such as high blood pressure and cholesterol.

[26] Hemp seed oil processed from hemp seeds can effectively slow down the increase of lipid peroxides in the body, thereby reducing TC, TG, and LDL levels, while increasing HDL, effectively reducing arteriosclerosis indicators. In addition, hemp seed oil can also alleviate the pathological conditions of arterial wall intima cells and smooth muscle cells, effectively inhibiting the formation of atherosclerotic plaques.

[27] . Hemp seed can not only be used to prevent and treat cardiovascular diseases, it can also reduce the risk of chronic diseases such as cancer, neurodegenerative diseases, lipid metabolism diseases, skin diseases and gastrointestinal diseases, and also protect the cardiovascular and cerebrovascular system. The antioxidant and antihypertensive peptides, sterols and their derivatives, and tocopherols contained in hemp seed can all affect lipid metabolism. In addition, hemp seed can also effectively treat glaucoma, cancer, constipation, hypertension and high cholesterol, promote immune regulation, improve skin diseases, and have anti-oxidation and anti-aging effects. Hemp seed has a high fat content. Excessive consumption may cause excessive burden on the liver, resulting in liver damage and symptoms such as hepatitis. In addition, hemp seed contains mushroom toxins. Excessive consumption may cause enterotoxins, abdominal pain, diarrhea, vomiting and other symptoms, and in severe cases may cause liver damage. Hemp seed contains choline, and excessive consumption may also cause choline poisoning, which can lead to coma in severe cases. Therefore, when eating hemp seed, you should pay attention to controlling the amount of consumption to avoid side effects.

[28] .

[0014] 1.3 Pueraria root

[0015] 1.3.1 Active ingredients of Pueraria root

[0016] Pueraria root is the dried root of the perennial leguminous plant Pueraria Lobata (Wild.) Ohwi or sweet kudzu vine, commonly known as wild kudzu. China is rich in kudzu root and its growing areas are mainly distributed in Henan, Hubei, Guangxi and Hunan. It tastes sweet and spicy, is cool in nature, and enters the lung and stomach meridians. It has the effects of enhancing yang energy, stopping diarrhea, relieving muscles and reducing fever. The book "Shennong's Herbal Classic" points out that kudzu root was originally used to treat "thirst, high fever, vomiting, various arthralgias, raising yin energy, and removing various toxins". It is a medicinal material that can be used as both medicine and food.

[29] . It is reported that the chemical components of Pueraria root mainly include alkaloids, isoflavones and polysaccharides, etc., which have significant therapeutic effects in sobering up, protecting the liver, lowering blood lipids, lowering blood sugar, protecting the myocardium and anti-oxidation. In addition, Pueraria root also contains nutrients such as dietary fiber, starch, mineral elements, and essential amino acids that the human body cannot synthesize. Modern pharmacological research has found that the active peptide components in Pueraria root have good functional properties and biological activity, and have the effects of improving cardiovascular and cerebrovascular diseases, anti-tumor, anti-virus, anti-free radicals, protecting the liver, and regulating immunity.

[30] As a natural plant-derived substance, Pueraria root has fewer side effects than modern synthetic drugs, which is in line with the most promising natural medicine industry.

[0017] 1.3.2 Pharmacological effects of Pueraria root

[0018] Pueraria root has pharmacological effects such as antipyretic, anti-inflammatory, anti-infective, anti-cancer, anti-atherosclerotic, lowering blood pressure, lowering blood sugar, lowering blood lipids, protecting the liver, preventing osteoporosis, and improving reproductive function.

[31] Studies have shown that Pueraria root has a protective effect on human liver damage and can be used as a potential therapeutic drug for chronic diseases of liver dysfunction and neurological deficits.

[32] Many studies have shown that Pueraria root has antioxidant effects, strong DPPH free radical scavenging activity, and can inhibit linoleic acid peroxidation and lipoxygenase activity.

[33] . In addition, Pueraria root can play an anti-diabetic and its complications role by improving insulin resistance, protecting pancreatic β-cell function, inhibiting inflammation, regulating autophagy, anti-oxidative stress, and affecting insulin and glucagon receptor signaling pathways. Wang et al. used a high-fat diet (HFD)-induced diabetic mouse model, exendin-4 (GLP-1R agonist) and metformin as positive controls to study the effects of puerarin in diabetic mice. The results showed that puerarin could improve blood glucose homeostasis in diabetic mice, and markers of new β-cell formation (insulin, PDX1, and Ngn3) were observed in the pancreatic ducts of diabetic mice treated with puerarin, while inducing the expression of insulin and PDX1, upregulating the expression of GL-1R and subsequently activating β-catenin and STAT3.

[34] The application of Pueraria root in the treatment of diabetes can effectively improve its insulin resistance, with few adverse reactions and significant effects.

[0019] 1.4 Coffee

[0020] 1.4.1 Types of Coffee

[0021] Coffee belongs to the Rubiaceae family and is a wild plant native to north-central Africa.

[35] . Coffee is a perennial evergreen shrub or small tree. There are many species of coffee, with more than 90 species in central and northern Africa. It is loved by people all over the world for its unique aroma and rich taste. Its small, medium and large varieties can be used for commercial cultivation, but the large variety is rarely used for commercial cultivation due to its poor quality. Small-grain coffee is the most popular type of coffee. It is characterized by long-lasting fragrance, overflowing fragrance, mellow taste, and more mellow taste than other varieties. It is deeply loved by European and American consumers. Its planting area is mainly distributed in southern Ethiopia. Among them, the most popular varieties are Catim, Arabica, Robusta, Typica and other varieties. Medium-grain coffee is produced in Congo. It has the characteristics of high yield and strong fragrance. It is not easy to be infected by diseases and beetles. Large-grain coffee, a strong, bitter-tasting coffee from Liberia, has the characteristics of deep roots, drought and wind resistance. Mature trees do not need shade, but are prone to disease. Due to its unique aroma and taste, it has a high value in the market and is favored by consumers. Small-grain, medium-grain and large-grain coffees all have their own unique characteristics and are suitable for different consumers. They are not only popular in China, but also occupy an important position in the international market.

[36] .

[0022] 98% of China's coffee production comes from Yunnan, where the main crop is small-grain coffee, which has a unique flavor of "strong but not bitter, fragrant but not strong, and slightly sour." With the improvement of people's living standards and the enhancement of health awareness, Yunnan small-grain coffee has been loved by more consumers, and more and more people have noticed its active ingredients and physiological functions.

[28] .

[0023] 1.4.2 Research on the composition and efficacy of coffee

[0024] Coffee is a non-alcoholic beverage made from coffee beans and is rich in a variety of beneficial organic substances and minerals.

[37] . The main components of coffee are caffeine, theobromine, theophylline, tannic acid, fat, etc. Caffeine (caffeine) is a special type of organic substance and a xanthine alkaloid. It is commonly found in coffee, cola, chocolate, tea and certain medicines. The main source of caffeine now is coffee. Caffeine is a compound with multiple functions. It is the most common diuretic and the most commonly used stimulant. It is widely used in many products, such as: coffee, tea, cola, soda water, chocolate, certain medicines and sports supplements. It can be used as a plant pesticide, as a drug with antipyretic, stimulating and antiviral effects, and even as a flavoring to make drinks and food more delicious. The molecular structure of caffeine is C8H 10 N4O2, colorless needle-shaped crystals, melting point is 235-238℃

[38] , low solubility, can only be dissolved in water, ethanol, ether and chloroform, but not in benzene. Caffeine is easily absorbed by the human body and has a strong cardiovascular stimulating effect, which can increase heart rate and blood pressure, thereby playing a role in refreshing the mind. However, caffeine is also a toxic substance. If consumed in excess, it can cause adverse reactions such as dizziness, insomnia, and palpitations. Oral LD50 in rats 50 192 mg / kg, generally considered safe

[39] . Caffeine is a central nervous system stimulant that can affect the activity of the human nervous system. At present, caffeine is widely used in various fields, especially in the cola and energy drink industries, but reports about it have also attracted attention and controversy. Caffeine is broken down into three primary metabolites in the liver: paraxanthine (84%) can promote the dissolution of lipids, thereby increasing glycerol and free fatty acids in the blood; theobromine (12%) can dilate blood vessels, increase urine volume, and play a role in improving physical health; theophylline (4%) can soothe bronchial smooth muscles and help treat asthma. Many studies at home and abroad have confirmed that drinking pure coffee will not only not harm the body, but can also be beneficial to health. Pure coffee refers to coffee without any other substances added. It has extremely low calories, and drinking in moderation can bring benefits to the body.

[32] For example, it can reduce the risk of cancer, protect cardiovascular system, enhance memory, prevent diabetes, help prolong life, etc.

[40] In addition, studies have found that caffeine can promote lipid metabolism in liver cells, thereby reducing the risk of fatty liver.

[41] 70% of diabetic and obese patients suffer from non-alcoholic fatty liver disease, but there is currently no specific treatment. However, patients who consume about 4 cups of coffee or tea a day, under the influence of caffeine, have a significantly lower incidence rate than those who consume less than 4 cups a day.

[42] .

[0025] There is no related report in the prior art about using hemp seed polypeptide, kudzu root polypeptide and coffee for treating non-alcoholic fatty liver disease.

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[0068] In order to solve the above problems, the present invention provides a kudzu coffee for improving non-alcoholic fatty liver disease and its application. The present invention uses a high-fat diet (HFD) to feed and establish a NAFLD mouse model. The kudzu coffee is used for intragastric gavage treatment, and the effects of the kudzu coffee on liver fat, blood lipids and other related metabolism are observed, and its efficacy and safety in treating non-alcoholic fatty liver disease are studied. The study found that through the active treatment of kudzu coffee, not only the burden on the liver is reduced, and the fatty liver lesions are improved, but also the lipid deposition in the body is reduced, which plays a good therapeutic role in improving non-alcoholic fatty liver disease. It provides an experimental basis for further exploring the effective material basis and mechanism of action for lowering blood lipids and protecting the liver.

[0069] In order to achieve the above object, the present invention provides the following technical solutions:

[0070] The present invention provides a kudzu coffee for improving non-alcoholic fatty liver disease, comprising the following components in parts by weight:

[0071] 0.5 parts of hemp seed polypeptide solution, 0.5 parts of kudzu root polypeptide solution and 9 parts of coffee;

[0072] The concentration of hemp seed polypeptide in the hemp seed polypeptide solution is 15 mg / mL;

[0073] The concentration of the Pueraria lobata polypeptide in the Pueraria lobata polypeptide solution is 5 mg / mL.

[0074] Preferably, the preparation method of the hemp seed polypeptide comprises the following steps:

[0075] 1) Mixing defatted hemp seed meal with water, adjusting the pH value to 8.5, performing magnetic stirring, and centrifuging for 20 minutes to obtain a supernatant;

[0076] The mass ratio of the defatted hemp seed meal to water is 1:12;

[0077] Use 1 mol / L NaOH solution to adjust pH value;

[0078] The magnetic stirring conditions include: temperature of 45°C, rotation speed of 800 rpm, and time of 1 h;

[0079] The centrifugal speed is 8000 rpm;

[0080] 2) adjusting the pH value of the supernatant obtained in step 1) to 4.5, allowing the supernatant to settle, and centrifuging for 15 minutes to obtain a precipitate, which is hemp seed protein;

[0081] The pH value of the supernatant was adjusted using 1 mol / L HCl solution;

[0082] The static sedimentation time is 5 minutes;

[0083] The centrifugal speed is 8000 rpm;

[0084] 3) mixing the hemp seed protein obtained in step 2) with water to obtain a protein solution;

[0085] The concentration of hemp seed protein in the protein solution is 0.05 g / mL;

[0086] 4) mixing the protein solution obtained in step 3) with alkaline protease, performing enzymolysis, and inactivating the enzyme to obtain an enzymolyzate;

[0087] The mass ratio of the hemp seed protein to the alkaline protease is 40:7;

[0088] The enzyme activity of the alkaline protease is 2000U / g;

[0089] The speed of the enzymatic hydrolysis is 200 rpm and the time is 12 h;

[0090] The enzyme inactivation temperature is 100°C and the time is 10 minutes;

[0091] 5) The pH value of the enzymolyzate obtained in step 4) is adjusted to be neutral, and the solution is centrifuged at 8000 rpm for 15 min to obtain a supernatant which is a hemp seed polypeptide solution. The concentration of the hemp seed polypeptide in the hemp seed polypeptide solution is 15 mg / mL.

[0092] Preferably, the method for preparing the Pueraria polypeptide comprises the following steps:

[0093] 1) mixing kudzu root powder with Tris-HCl buffer, extracting and soaking overnight to obtain an extract;

[0094] The mass ratio of the kudzu root powder to the volume ratio of the Tris-HCl buffer solution is 2kg:20L;

[0095] The concentration of the Tris-HCl buffer is 50 mmol / L, and the pH value is 9.0;

[0096] The extraction conditions include: temperature of 50°C and time of 2h;

[0097] 2) centrifuging and filtering the extract obtained in step 1) to obtain a filtrate, allowing the filtrate to stand and centrifuge to obtain a precipitate, and freeze-drying the precipitate to obtain pueraria protein;

[0098] The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 10 min;

[0099] The standing time is 2h;

[0100] The freeze-drying conditions include: temperature of -70°C and time of 48h;

[0101] 3) mixing the pueraria protein obtained in step 2) with sodium borate buffer and alkaline protease, performing enzymolysis and inactivating the enzyme to obtain an enzymolysate;

[0102] The mass ratio of the pueraria protein to the volume of the sodium borate buffer and the alkaline protease is 25 g:100 mL:4 g;

[0103] The pH value of the sodium borate buffer is 9.0;

[0104] The enzymatic activity of the alkaline protease is 3500U / g;

[0105] The conditions of the enzymatic hydrolysis include: time of 2 h, temperature of 61° C., and pH of 9.0;

[0106] The enzyme inactivation conditions include: temperature of 100°C and time of 10 min;

[0107] 4) mixing the enzymatic hydrolysate obtained in step 3) with a trichloroacetic acid solution, adjusting the pH value to be neutral, and then standing, centrifuging to obtain a supernatant as a pueraria polypeptide solution, wherein the concentration of the pueraria polypeptide in the pueraria polypeptide solution is 5 mg / mL;

[0108] The volume ratio of the trichloroacetic acid solution to the sodium borate buffer solution is 1:1, and the mass percentage of the trichloroacetic acid solution is 15%;

[0109] The standing time is 0.5h;

[0110] The centrifugal conditions include: a rotation speed of 4000 rpm and a time of 10 min.

[0111] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a medicine for treating non-alcoholic fatty liver disease.

[0112] The present invention also provides the use of the Kudzu coffee described in the above technical solution in the preparation of a drug for reducing lipid deposition in non-alcoholic fatty liver and / or liver fat in non-alcoholic fatty liver.

[0113] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing inflammatory damage of non-alcoholic fatty liver disease.

[0114] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing the liver coefficient of non-alcoholic fatty liver.

[0115] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing the triglyceride and / or cholesterol content in non-alcoholic fatty liver.

[0116] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for increasing the high-density lipoprotein content in non-alcoholic fatty liver.

[0117] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing alanine aminotransferase in non-alcoholic fatty liver and / or reducing aspartate aminotransferase in non-alcoholic fatty liver.

[0118] The mechanism of the kudzu coffee provided by the present invention in treating non-alcoholic fatty liver disease is:

[0119] The present invention provides the kudzu coffee described in the above technical solution, which improves the degree of liver function damage, thereby alleviating the symptoms of non-alcoholic fatty liver disease.

[0120] The present invention provides the Kudzu coffee described in the above technical solution, which improves the degree of liver fibrosis and thus alleviates the symptoms of non-alcoholic fatty liver disease.

[0121] The present invention provides the kudzu coffee described in the above technical solution, which improves lipid metabolism disorder and thus alleviates the symptoms of non-alcoholic fatty liver.

[0122] The present invention provides the Kudzu coffee described in the above technical solution, which alleviates the symptoms of non-alcoholic fatty liver by improving the inflammatory response.

[0123] Beneficial effects:

[0124] After the mice were treated with kudzu coffee by gavage in the present invention, it was observed that the mice's coat color, spirit, and activity were all well improved. In the other two groups, the blank group mice were slender, with bright coat color and good activity. After the model group was fed a high-fat diet for 4 consecutive weeks, the mice began to have gray coat color, less activity, lack of spirit, greasy and rough fur, hypertrophy, body weight, body weight growth rate, liver weight, and liver index were significantly higher than those of the blank group, and serum TG and TC levels also increased significantly. It shows that the high-fat diet-induced NAFLD mouse model was successfully constructed. After 7 consecutive weeks of treatment, according to the appearance of the liver, liver tissue pathology observations found that the kudzu coffee mixture effectively reduced lipid deposition and liver fat, and improved liver fatty lesions. It can be seen from the experimental results that kudzu coffee has a significant lipid-lowering effect, can effectively reduce liver fat particles, reduce inflammatory damage, improve liver tissue pathological lesions, and alleviate liver tissue damage in NAFLD mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0126] Figure 1 The changes of body weight and Lee's index of mice in each group. Note: Compared with the model group, * P<0.05, ** P<0.01; compared with the model group and the Ge Ma coffee group, # P<0.05, ## P < 0.01;

[0127] Figure 2 is the liver index, Note: Comparison between blank group and model group * P<0.05, ** P<0.01; compared with the model group and the Ge Ma coffee group, # P<0.05, ## P < 0.01;

[0128] Figure 3 Fasting blood glucose and blood lipid levels. Note: Comparison between blank group and model group * P<0.05, ** P<0.01; compared with the model group and the Ge Ma coffee group, # P<0.05, ## P < 0.01;

[0129] Figure 4 are biochemical indicators of alanine aminotransferase and aspartate aminotransferase. Note: Compared with the blank group and the model group, * P<0.05, ** P<0.01; compared with the model group and the Ge Ma coffee group, #P<0.05, ## P < 0.01;

[0130] Figure 5 The morphological characteristics and pathological morphology of mouse liver (HE×100, Oil Red×100). Note: The blank group was compared with the model group. * P<0.05, ** P<0.01; compared with the model group and the Ge Ma coffee group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0131] The present invention provides a kudzu coffee for improving non-alcoholic fatty liver disease, comprising the following components by weight: 0.5 parts of a hemp seed polypeptide solution, 0.5 parts of a kudzu root polypeptide solution and 9 parts of coffee; the concentration of the hemp seed polypeptide in the hemp seed polypeptide solution is 15 mg / mL; the concentration of the kudzu root polypeptide in the kudzu root polypeptide solution is 5 mg / mL. In the present invention, the hemp seed polypeptide and the kudzu root polypeptide are both bioactive peptides, have strong bioactivity, and can be effectively absorbed and utilized by the human body. Such bioactive peptides have the effect of promoting metabolism, can accelerate the oxidation of substances, and improve the basal metabolic rate. Certain components in coffee can help the body discharge waste and toxins in the body. The hemp seed polypeptide, the kudzu root polypeptide and coffee are used together to promote the detoxification and liver protection effects of coffee. Coffee can promote the excitement of the nervous system. If the agonists hemp seed polypeptide and the kudzu root polypeptide are used in excessive amounts, the stimulation effect will be enhanced, and problems such as accelerated heartbeat and insomnia will occur. Therefore, in the present invention, 0.5 parts of hemp seed polypeptide solution, 0.5 parts of kudzu root polypeptide solution and 9 parts of coffee are selected to prepare kudzu coffee.

[0132] In the present invention, the preparation of the hemp seed polypeptide adopts an enzymatic hydrolysis method (Chen Xia, Xiao Xiao, Wang Peng, et al. Analysis of the quality characteristics of fermented milk with added hemp seed meal protein peptides [J]. Modern Food Science and Technology, 2021, 37 (08): 160-167.), the polypeptide prepared by this method has a small molecular weight and strong activity and diversity, and is widely used in polypeptide preparation. The specific preparation steps are as follows: the dried and crushed defatted hemp seed meal is mixed with water at a ratio of 1:12 (W / W), the pH value of the solution is adjusted to 8.5 with 1mol / LNaOH solution, and it is placed at 45°C and 800r / min on a constant temperature magnetic stirrer for 1h. After stirring, centrifuge at 8000r / min for 20min, take the supernatant, and adjust the pH of the solution to 4.5 with 1mol / L HCl solution. Let it stand and settle, centrifuge at 8000r / min for 15min, take the precipitate, wash it with distilled water until it is neutral, and freeze-dry it to obtain hemp seed meal protein. The hemp seed meal protein of 40g freeze-dried is mixed with the protein solution that concentration is 0.05g / mL with deionized water, add 7g alkaline protease (enzyme activity: 2000U / g), mix, be adjusted to optimum enzymolysis temperature and pH, be placed on constant temperature magnetic stirrer, with 200r / min oscillating reaction 12h, then continue enzyme deactivation 10min in boiling water.Be cooled to room temperature, solution is adjusted to neutrality, centrifugal 15min under 8000r / min, collect supernatant, promptly obtain 15mg / mL hemp seed polypeptide solution.In the present invention, the effect of described hemp seed polypeptide is anti-oxidation, blood pressure lowering, blood sugar lowering, immune regulation etc.

[0133] In the present invention, the preparation of the pueraria polypeptide also adopts the enzymatic hydrolysis method (Zhang Linsong, Xu Weidong, Shi Jiwei, et al. Study on the therapeutic effect of puerarin and pueraria polypeptide on alcoholic liver injury in mice [J]. Jiangsu Traditional Chinese Medicine, 2018, 50(02): 76-78.). The specific steps are as follows: weigh 2kg of pueraria powder and place it in a container, add 20000mL of 50mmol / L pH=9 Tris-HCl buffer, stir thoroughly, then extract in a 50℃ water bath for 2h and soak overnight; take the extract and centrifuge at 5000r / min for 10min, filter the supernatant, combine the filtrate, adjust the pH to 3 with concentrated hydrochloric acid, let it stand for 2h, centrifuge at 5000r / min for 10min, discard the supernatant, combine the precipitate, freeze it at -70℃ for 48h, and freeze-dry it in vacuum to obtain pueraria protein powder, which is stored in a desiccator for later use. Take the pueraria protein powder and wash it with anhydrous ethanol, and dry it naturally for later use. 25g of the above-mentioned kudzu root protein powder is placed in a 250mL beaker, 100mL of sodium borate buffer solution adjusted to pH 9.0 with 0.5mol / L hydrochloric acid or 0.5mol / L sodium hydroxide is added, stirred, placed in a 50°C water bath and left to stand for 20min to fully dissolve the protein, 4g of alkaline protease (enzyme activity: 3500U / g) is added, then placed in a water bath (temperature is 61°C) and reacted for 2h, and 0.1mol / L sodium hydroxide is used to maintain pH unchanged during the reaction. After the reaction is completed, a boiling water bath is used for 10min to inactivate alkaline protease, the water bath is cooled to room temperature, 15% trichloroacetic acid 100mL is added, stirred, and pH is adjusted to 7.0 with 0.5mol / L hydrochloric acid or 0.5mol / L sodium hydroxide, left to stand for half an hour, 4000r / min was centrifuged for 10min, and supernatant was taken to obtain a 5mg / mL kudzu root polypeptide solution. In the present invention, the kudzu root polypeptide has the effect of protecting the liver. In the present invention, the coffee is preferably coffee powder, which can stimulate the central nervous system, accelerate the body's metabolism, and stimulate the metabolism and detoxification functions of the liver, reduce the burden on the liver, and has a liver-protecting effect.

[0134] The present invention has no special limitation on the preparation method of the coffee powder. The coffee powder can be prepared according to conventional methods in the art, such as grinding coffee beans in a grinder, boiling in a constant temperature water bath, coarse filtering with gauze, suction filtering, and vacuum low-temperature drying / spray drying.

[0135] In the present invention, the preparation method of the kudzu coffee preferably comprises the following steps: mixing the hemp seed polypeptide solution, kudzu root polypeptide solution and coffee, adding distilled water 10 times the volume of the total mass of the drug, and then placing it in a 50°C water bath and heating and stirring for 10 minutes to dissolve it, thereby obtaining the kudzu coffee. In the present invention, the volume ratio of the total mass of the kudzu root polypeptide solution, the hemp seed polypeptide solution and coffee to distilled water is 10g:100mL. The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for treating non-alcoholic fatty liver disease. The present invention does not specifically limit the dosage form and preparation method of the drug, and those skilled in the art can use conventional methods.

[0136] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing lipid deposition in non-alcoholic fatty liver. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0137] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing liver fat in non-alcoholic fatty liver. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0138] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing non-alcoholic fatty liver inflammation damage. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0139] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing the liver coefficient of non-alcoholic fatty liver. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0140] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing triglyceride and / or cholesterol content in non-alcoholic fatty liver. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0141] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for increasing the content of high-density lipoprotein in non-alcoholic fatty liver. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can adopt conventional methods.

[0142] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing non-alcoholic fatty liver alanine aminotransferase. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can use conventional methods.

[0143] The present invention also provides the use of the kudzu coffee described in the above technical solution in the preparation of a drug for reducing non-alcoholic fatty liver aspartate aminotransferase. The present invention has no special limitation on the dosage form and preparation method of the drug, and those skilled in the art can use conventional methods.

[0144] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0145] Example 1

[0146] 1. Materials and Operation

[0147] 1.1 Experimental Materials

[0148] 1.1.1 Test materials

[0149] The experiment requires 15 C57BL / 6J mice, SPF grade, 6-week-old males. The initial weight range of each experimental group is 15-18g, purchased from Beijing Huafukang Biotechnology Co., Ltd., experimental animal production license: SCXK (Beijing) 2019-0008. According to relevant animal standards at home and abroad, they are raised in SPF animal breeding rooms, free to move, drink urban drinking water sterilized at 121℃ for 20 minutes, and feed with customized feed. The research team adds it according to the experimental needs. Modeling for 0-12 weeks, and drug treatment for 5-10 weeks.

[0150] Other materials required for the experiment: hemp seed peptide solution, the concentration of hemp seed peptide in the hemp seed peptide solution is 15 mg / mL (Chen Xia, Xiao Xiao, Wang Peng, et al. Analysis of the quality characteristics of fermented milk with hemp seed meal protein peptide [J]. Modern Food Science and Technology, 2021, 37(08): 160-167.); Pueraria peptide, the concentration of Pueraria peptide in the Pueraria peptide solution is 5 mg / mL (Zhang Linsong, Xu Weidong, Shi Jiwei, et al. Study on the therapeutic effect of puerarin and Pueraria peptide on alcoholic liver injury in mice [J]. Jiangsu Traditional Chinese Medicine, 2018, 50(02): 76-78.); coffee powder. Mouse wood shavings bedding, mouse basic feed and high-fat feed (20% protein + 20% carbohydrate + 60% fat) for fatty liver modeling. High-fat feed needs to be refrigerated at 4°C during use, and the rest should be frozen and stored at -20°C. The palatability of high-fat feed is slightly lower than that of ordinary feed, and animals are prone to anorexia and low feed intake, and it has a bad odor when left at room temperature. Gradual food replacement and fasting methods can be used, and feed can be added in small amounts and multiple times.

[0151] 1.2 Experimental procedures

[0152] 1.2.1 Preparation of intragastric gavage reagent

[0153] Preparation of Kudzu coffee intragastric administration reagent: Take 0.5g of hemp seed polypeptide, 0.5g of kudzu polypeptide, and 9g of coffee powder. Coffee powder preparation process: Select coffee beans picked in the mature season and remove immature, overripe or damaged fruits. Then put the fruits into the peeling machine to remove the peels to obtain coffee beans with pectin. Then put the coffee beans into the water tank for fermentation, usually for 12-36 hours. During the fermentation process, the temperature and humidity in the fermentation box should be closely monitored. Generally speaking, the temperature is maintained between 25-30℃ and the humidity is maintained between 80-90%. After the fermentation is completed, put the fermented coffee beans into the water tank, and then add water to stir thoroughly to separate the peel and pulp from the coffee beans. Then, rinse the coffee beans with clean water again to thoroughly wash the pectin and impurities. Finally, dry them. They can be dried in the sun in the sunning field or dried by machine to reduce the moisture content of the coffee beans to 10%-12%. The coffee beans are crushed by grinding equipment to obtain ground coffee powder, and the ground coffee bean powder is continuously extracted by high-pressure closed multi-tanks to obtain an extract. Usually, the extraction temperature should be between 90-95°C, the pressure should be 9 bar, and the time should be controlled between 25-40 seconds. The coffee extract is concentrated by R0 reverse osmosis membrane to obtain a coffee concentrate, the concentration of which is 10% (coffee concentration (%) = [total dissolved solids (g) / actual liquid weight (g)] * 100%, that is, the total solid coffee is 20 grams, the actual liquid weight is 200 grams, and the concentration is 10%). A certain weight of coffee concentrate is evenly spread on the freeze-drying plate and quickly frozen in a cold storage until it is completely frozen. The quick-frozen coffee concentrate is placed in a vacuum drying furnace, heated and dried under certain vacuum conditions and under the drying curve to obtain a finished coffee powder. The horizontal axis of the drying curve is time, and the vertical axis is temperature. The drying curve is: 20℃5min, 20℃2h, 30℃0.5h, 30℃3h, 50℃2h, 50℃5h, 65℃1h, 65℃10h. After weighing, mix the hemp seed polypeptide, kudzu polypeptide, and coffee powder, add 10 times the volume of distilled water of the total mass of the drug, and place it in a 50℃ water bath for heating and stirring for 10min to dissolve it to prepare the required reagent. After the configuration is completed, transfer it to a large centrifuge tube and indicate the name of the drug, store it in a -20℃ refrigerator, and take it out to room temperature before use when needed, or prepare it and use it now.

[0154] 1.2.2 NAFLD mouse model establishment and grouping and drug administration

[0155] Fifteen male C57 mice were divided into three groups using the average weight method, with 5 mice in each group. They were blank control group, model group, and kudzu coffee treatment group. There was no statistical difference in weight between the mouse groups. During the experiment, the blank group was fed with ordinary feed throughout the experiment, the model group was fed with high-fat feed, and the kudzu coffee treatment group was fed with high-fat feed and gavage treatment began in the 5th week. The experimental period was 12 weeks in total. The dosage of the kudzu coffee group mice was 0.1ml / 10g, and the blank group and the model group were gavaged with an equal dose of distilled water once a day at the same time. At the beginning of modeling, the body weight was weighed once a week, the body length (the distance from the tip of the nose to the outer edge of the anus) was measured once, and the Lee's index was calculated (Lee's index: body weight (g) ^ (1 / 3) x10 / body length cm). The same amount of feed was put in every day, and the weekly food intake was recorded. Statistical analysis of changes in body weight, body length, etc. from modeling to treatment. Observe the mental state, behavioral performance, food intake, water intake, and death of the mice, and keep experimental records.

[0156] 1.2.3 Index determination

[0157] After the last oral gavage treatment in the 12th week, the mice were fasted but not watered for 24 hours, and their stomachs were emptied and their body weight was weighed and recorded. After anesthesia with 1% sodium pentobarbital, the eyeballs were removed and blood was collected. After the blood was allowed to stand for 2 hours, it was centrifuged at 3000r / min and 4℃ for 10 minutes, and the supernatant was taken. The contents of serum, total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by an automatic biochemical analyzer in strict accordance with the instructions of the reagents. The mice were dissected, and the liver tissue of the mice was removed on the ice. The color, size and thickness of the liver tissue were observed, and photos were taken for comparison. Then the peeled liver was weighed and the organ coefficient was calculated. The formula for calculating the organ coefficient is: organ coefficient (%) = organ weight / body weight × 100.

[0158] 1.3 Data processing

[0159] After the experimental data were entered into the Excel table and confirmed to be correct, the data obtained from the application study were analyzed using SPSS21.0 software, and the independent sample t test was used between the groups. P<0.05 was statistically significant, and P<0.01 was extremely significant. GraphPadprism 8.0.2.263 software was used for graphical data analysis.

[0160] 1.3.1 Changes in body weight and Lee's index of mice in each group

[0161] Lee's index can better reflect the obesity degree of rats / mice. The Lee's index is obtained by calculating the formula and plotting it as follows: Figure 1 As shown in A, there was a significant difference between the blank group and the model group (P<0.01), but there was no significant difference in the Lee's index between the model group and the Ge Ma coffee group; Figure 1 There was no statistical difference in body weight among mice in the B group before modeling. The body weight of mice in the blank group, model group and kudzu coffee group showed an upward trend from the 3rd week. From the 4th week, there was a significant difference in body weight between the blank group and the model group (P<0.05, P<0.01). Drug administration began in the 5th week. By the 10th week, the body weight of mice in the kudzu coffee group was significantly reduced compared with the model group (P<0.05). The body weight growth rate was as follows: Figure 1 In Figure C, there was a significant difference between the blank group and the model group at the 4th week (P<0.05, P<0.01). At the 10th week, there was a significant difference in the weight growth rate of the mice in the kudzu coffee group compared with the model group (P<0.05).

[0162] 1.3.2 Liver index

[0163] The organ mass of animals is an important indicator of biological characteristics, which can clarify the degree of development of various organs in the animal body to a certain extent. In general, organ weight is proportional to body weight, but organ mass is affected by many factors, such as the influence of internal factors (breed, gender, weight and age, etc.) and experimental factors (season and feeding conditions, etc.). When the animal body is damaged, the weight of its internal organs will change, and the organ index will also change accordingly. If the organ index becomes larger, it may indicate that the organ has pathological lesions such as congestion, edema, hyperplasia, hypertrophy, etc.; if the organ index becomes smaller, it may indicate that the organ has atrophied or other damage has occurred. From Figure 2 It can be seen that the liver coefficient of mice in the model group was significantly increased compared with that in the blank group (P < 0.01); compared with the model group, the liver coefficient of mice in the Ge Ma coffee group was decreased (P < 0.05).

[0164] 1.3.3 Effects of Kudzu Coffee on Blood Glucose and Lipid Levels in Mice with Non-alcoholic Fatty Liver Disease

[0165] from Figure 3 It can be seen that the serum glucose content of each group of mice was measured and the data was analyzed graphically. There was no significant difference in blood glucose between the blank group mice and the model group mice, and between the model group mice and the Ge Ma coffee group mice.

[0166] Blood lipids are lipid substances in plasma, including TC, TG, HDL-C and LDL-C, which can provide energy and store energy for the body and maintain the structure and function of biological membranes. Dyslipidemia and fatty degeneration of hepatocytes are related to non-alcoholic fatty liver disease. Figure 3 It can be seen that compared with the blank group, the triglyceride and low-density lipoprotein levels in the serum of the model group mice were significantly increased (P<0.01), the cholesterol level was also significantly increased (P<0.05), and the high-density lipoprotein level was significantly decreased (P<0.01). Compared with the model group, the triglyceride in the serum of the kudzu coffee group mice was significantly different (P<0.01), and the cholesterol and high-density lipoprotein showed significant differences (P<0.05). This shows that the non-alcoholic fatty liver mouse model induced by a high-fat diet was successfully established, and the treatment of kudzu coffee also had a certain effect.

[0167] 1.3.4 Effects of Kudzu Coffee on AST and ALT in Mice with Nonalcoholic Fatty Liver Disease

[0168] AST and ALT are important indicators of liver function testing. They are transaminases present in liver cells. When liver cells are damaged, their metabolism becomes abnormal. Therefore, the level of AST and ALT can be used to assess the degree of liver cell damage. In the study of chronic hepatobiliary diseases, the damage to liver cells can be better understood by measuring the levels of AST and ALT. Figure 4 It can be seen that by providing mice with a high-fat diet to simulate chronic hepatobiliary diseases, after 12 weeks of experiments, it was found that the alanine aminotransferase content in the model group was significantly increased compared with the blank group (P<0.05), indicating that the high-fat diet successfully induced liver cell damage in mice. At the same time, this also proves that AST and ALT are effective indicators for measuring chronic hepatobiliary diseases.

[0169] 1.3.5 Observation of liver appearance and liver tissue pathology of mice in each group

[0170] The liver appearance and liver tissue of mice were examined by HE staining and Oil Red O staining. Figure 5As shown. The livers of mice in the blank group were normal in size, reddish-brown in appearance, soft in texture, with sharp edges and smooth cut surfaces; while the livers of mice in the model group were significantly enlarged, with yellow fat deposits visible in appearance, hard in texture, blunt and thick edges, and greasy cut surfaces; the liver volume, appearance, and texture of mice in the kudzu coffee group were between the model group and the blank group. In the HE staining results, the liver tissue cells of the blank group had clear boundaries and were arranged neatly, without fat droplets and vacuoles, and the cell nuclei were normal in size. In the model group, the liver cells were enlarged, the liver tissue was chaotic, the boundaries were unclear, larger lipid droplets were visible, and there were more fat vacuoles in the intercellular space, which was significantly different from the blank group (P<0.01); the kudzu coffee group had significantly fewer fat vacuoles in the liver tissue than the model group (P<0.05), and the boundaries were clearer than the model group, which significantly reduced the degree of fatty degeneration of the mouse liver. Oil red O staining results: The dye made the fat droplets in the tissue orange-red to bright red, and the cell nuclei were blue-purple. The untreated model group contained a large number of lipid droplets clustered together, which was significantly different from the blank group (P<0.01). After treatment, the fat oil droplets in the liver tissue of the Ge Ma coffee group were significantly reduced (P<0.05), only a small amount of inflammatory cells infiltrated in the hepatocytes, and the cell morphology was significantly improved.

[0171] 2. Conclusion and Outlook

[0172] 2.1 Conclusion

[0173] After the mice were treated with kudzu coffee by gavage in the present invention, it was observed that the mice's coat color, spirit, and activity were all well improved. In the other two groups, the blank group mice were slender, with bright coat color and good activity. After the model group was fed a high-fat diet for 4 consecutive weeks, the mice began to have gray coat color, less activity, lack of spirit, greasy and rough fur, hypertrophy, body weight, body weight growth rate, liver weight, and liver index were significantly higher than those of the blank group, and serum TG and TC levels also increased significantly. It shows that the high-fat diet-induced NAFLD mouse model was successfully constructed. After 7 consecutive weeks of treatment, according to the appearance of the liver, liver tissue pathology observations found that the kudzu coffee mixture effectively reduced lipid deposition and liver fat, and improved liver fatty lesions. It can be seen from the experimental results that kudzu coffee has a significant lipid-lowering effect, can effectively reduce liver fat particles, reduce inflammatory damage, improve liver tissue pathological lesions, and alleviate liver tissue damage in NAFLD mice.

[0174] 2.2 Outlook

[0175] In recent years, NAFLD, as a common and frequent liver disease in clinical practice, has posed a huge threat to the health of patients, and its incidence rate has also shown an increasing trend year by year. It is generally believed that NAFLD is related to factors such as insulin resistance and genetics. Recent studies have found that in addition to factors such as insulin resistance, obesity, and type 2 diabetes, the accumulation of free cholesterol in the liver is also closely related to the pathogenesis of non-alcoholic fatty liver disease. Clinically, this disease seriously threatens the health of patients, and there is currently no effective treatment that can reverse the progression of NAFLD. Therefore, it is particularly important to explore new functional foods or drugs to improve NAFLD. The drug treatment of NAFLD is generally divided into Western medicine and traditional Chinese medicine. Western medicine can be treated with liver-protecting and lipid-lowering drugs. There are many types of liver-protecting and lipid-lowering drugs, but due to the lack of rigorous experiments, it is difficult to evaluate their exact efficacy, and there may be large side effects or unsatisfactory treatment effects. Therefore, caution should be exercised when using drugs to avoid drug abuse. The principle of high effectiveness and few side effects should be followed to ensure the safe and effective use of drugs to achieve the best therapeutic effect. Traditional Chinese medicine can be used for dialectical treatment according to the main symptoms. Starting from the holistic concept and the concept of dialectical treatment, prescriptions such as liver-clearing and gallbladder-promoting, liver-soothing and fat-removing are used to alleviate symptoms. However, the pharmacological mechanism of action has not kept pace with modern medicine. In addition, there are many traditional Chinese medicines for the treatment of NAFLD, and most of the drugs still need further clinical research, so traditional Chinese medicine cannot be widely used. In the fatty liver experiment, animals (mice) fed with a high-fat diet can replicate the tissue lesions and pathogenesis in human non-alcoholic fatty liver. Long-term feeding of a high-fat diet will cause mice to become obese and have fatty degeneration of the liver. In this experiment, Kudzu coffee was used to treat NAFLD mice by gavage to investigate the protective effect of Kudzu coffee on the liver. ALT and AST are important evaluation indicators of liver function. The experimental results showed that Kudzu coffee can effectively improve the levels of ALT and AST, reduce cholesterol and triglycerides, indicating that it may have an improvement effect on liver damage. Kudzu coffee contains a large number of active ingredients, has rich pharmacological effects, and has a good application prospect. As our understanding of the pathogenesis of NAFLD continues to deepen, new drugs and treatments will continue to emerge. In future research, if we can understand the pathogenesis of NAFLD and purify the effective ingredients of Chinese medicinal materials or natural medicines, we will be able to further achieve the goal of improving efficacy.

[0176] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Kudzu coffee for improving non-alcoholic fatty liver disease, characterized in that: The composition comprises the following components in parts by weight: 0.5 parts of hemp seed polypeptide solution, 0.5 parts of kudzu root polypeptide solution and 9 parts of coffee; The concentration of hemp seed polypeptide in the hemp seed polypeptide solution is 15 mg / mL; The concentration of the Pueraria lobata polypeptide in the Pueraria lobata polypeptide solution is 5 mg / mL.

2. The Kudzu coffee according to claim 1, characterized in that The preparation method of the hemp seed polypeptide comprises the following steps: 1) Mixing defatted hemp seed meal with water, adjusting the pH value to 8.5, performing magnetic stirring, and centrifuging for 20 minutes to obtain a supernatant; The mass ratio of the defatted hemp seed meal to water is 1:12; Use 1 mol / L NaOH solution to adjust pH value; The magnetic stirring conditions include: temperature of 45°C, rotation speed of 800 rpm, and time of 1 h; The centrifugal speed is 8000 rpm; 2) adjusting the pH value of the supernatant obtained in step 1) to 4.5, allowing the supernatant to settle, and centrifuging for 15 minutes to obtain a precipitate, which is hemp seed protein; The pH value of the supernatant was adjusted using 1 mol / L HCl solution; The static sedimentation time is 5 minutes; The centrifugal speed is 8000 rpm; 3) mixing the hemp seed protein obtained in step 2) with water to obtain a protein solution; The concentration of hemp seed protein in the protein solution is 0.05 g / mL; 4) mixing the protein solution obtained in step 3) with alkaline protease, performing enzymolysis, and inactivating the enzyme to obtain an enzymolyzate; The mass ratio of the hemp seed protein to the alkaline protease is 40:7; The enzyme activity of the alkaline protease is 2000U / g; The speed of the enzymatic hydrolysis is 200 rpm and the time is 12 h; The enzyme inactivation temperature is 100°C and the time is 10 minutes; 5) The pH value of the enzymolyzate obtained in step 4) is adjusted to be neutral, and the solution is centrifuged at 8000 rpm for 15 min to obtain a supernatant which is a hemp seed polypeptide solution. The concentration of the hemp seed polypeptide in the hemp seed polypeptide solution is 15 mg / mL.

3. The Kudzu coffee according to claim 1, characterized in that The preparation method of the Pueraria polypeptide comprises the following steps: 1) mixing kudzu root powder with Tris-HCl buffer, extracting and soaking overnight to obtain an extract; The mass ratio of the kudzu root powder to the volume ratio of the Tris-HCl buffer solution is 2kg:20L; The concentration of the Tris-HCl buffer is 50 mmol / L, and the pH value is 9.0; The extraction conditions include: temperature of 50°C and time of 2h; 2) centrifuging and filtering the extract obtained in step 1) to obtain a filtrate, allowing the filtrate to stand and centrifuge to obtain a precipitate, and freeze-drying the precipitate to obtain pueraria protein; The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 10 min; The standing time is 2h; The freeze-drying conditions include: temperature of -70°C and time of 48h; 3) mixing the pueraria protein obtained in step 2) with sodium borate buffer and alkaline protease, performing enzymolysis and inactivating the enzyme to obtain an enzymolysate; The mass ratio of the pueraria protein to the volume of the sodium borate buffer and the alkaline protease is 25 g:100 mL:4 g; The pH value of the sodium borate buffer is 9.0; The enzymatic activity of the alkaline protease is 3500U / g; The conditions of the enzymatic hydrolysis include: time of 2 h, temperature of 61° C., and pH of 9.0; The enzyme inactivation conditions include: temperature of 100°C and time of 10 min; 4) mixing the enzymatic hydrolysate obtained in step 3) with a trichloroacetic acid solution, adjusting the pH value to be neutral, and then standing, centrifuging to obtain a supernatant as a pueraria polypeptide solution, wherein the concentration of the pueraria polypeptide in the pueraria polypeptide solution is 5 mg / mL; The volume ratio of the trichloroacetic acid solution to the sodium borate buffer solution is 1:1, and the mass percentage of the trichloroacetic acid solution is 15%; The standing time is 0.5h; The centrifugal conditions include: a rotation speed of 4000 rpm and a time of 10 min.

4. Use of the Kudzu coffee according to claim 1 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

5. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for reducing lipid deposition in non-alcoholic fatty liver and / or liver fat in non-alcoholic fatty liver.

6. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for reducing inflammatory damage in non-alcoholic fatty liver disease.

7. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for reducing the liver coefficient of non-alcoholic fatty liver disease.

8. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for reducing the content of triglycerides and / or cholesterol in non-alcoholic fatty liver.

9. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for increasing the content of high-density lipoprotein in non-alcoholic fatty liver.

10. Use of the Kudzu coffee according to claim 1 in the preparation of a drug for reducing alanine aminotransferase and / or aspartate aminotransferase in non-alcoholic fatty liver.

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